First Report of Anthracnose on Dioscorea opposita Caused by Colletotrichum siamense in Guangdong province in China.

Yam is the world's fourth most important tuber crop, after cassava, potato, and sweet potato in the world, the cultivation area of yam from the Food and Agriculture Organization of the United Nations Statistics Division database (FAOSTAT) is about 8,831,037 ha in 2020. Chinese yam (Dioscorea opposita Thunb.) is an economically important root crop throughout China due to its high economic and medicinal value. South China including Guangdong and Guangxi provinces is one of the important production districts of Chinese yam with economic value. A disease affecting the leaves was observed on yam leaves in August 2021 with an incidence of 20 to 90% in Guangdong and Guangxi provinces. Symptoms start as pinpoint lesions on yam leaves which enlarged to oval spots and large irregular spots. The spots were brown and surrounded by a chlorotic halo with sunken cavities, which are typical symptoms of anthracnose. To identify the causal agent, 9 symptomatic leaves from 3 different districts were collected in Guangdong and Guangxi provinces. Leaf samples were disinfested with 1% NaOCl for 3 min, and cultured on potato dextrose agar (PDA) at 28 °C for 3 days week. 9 single-spore isolates were recovered from each PDA medium. Colonies on PDA were grayish white with bright orange conidial spore masses. Fungal mycelia were hyaline, septate, and branched. Conidia were born on a long conidiogenous cell, straight, hyaline and cylindrical with rounded ends, 5.3 to 6.8×15.2 to 21.3μm (n = 50). Appressoria were dark, smooth-walled, oval in shape. The isolates were morphologically identified as Colletotrichum sp. (Weir et al. 2012). 3 strains were used for the pathogenicity test, 5 plants at creeping stage were inoculated with each isolate separately and 3-5 leaves of each plant were inoculated. Fresh wounds were made with a sterile needle on the healthy surface of yam leaves and each leaf was covered with a piece of cotton drenched with conidial suspension (106 conidia/mL) from each isolate. Control seedlings were inoculated identically except sterile water was used. Inoculated plants were placed in a moisturizing light incubator at 25℃ and 80% humidity under a 12-h light/dark cycle for 7 days and examined daily to monitor disease symptom development. Small round brown spots were observed at the inoculation sites 3 days after the inoculation and eventually became large brown lesions. No symptoms wre observed in the water-inoculated plants. A Colletotrichum sp. strain based on morphology was reisolated from inoculated leaves, fulfilling Koch's postulates. For molecular identification, the direct colony PCR method (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS) region of ribosomal DNA, calmodulin (Cal), tubulin (Tub) and Apmat loci of three isolates using primer pairs of ITS4/ITS5, CL1C/CL2C, T1/T2 and AM-F/AM-R (Sharma et al. 2015). A phylogenetic tree derived from a maximum likelihood analysis of a concatenated alignment of ITS, Cal, Tub and ApMAT sequences was created. The accession numbers of the three isolates YamZJCS, YamSXCS and YamYLCS used in this study were OP128056-OP128058 for ITS, OP128059-OP128061 for ApMAT，OP128062-OP128064 for Cal and OP128065-OP128067 for Tub. The sequences of the 3 isolates were aligned with related species of Colletotrichum (Sharma et al. 2015). Analyses based on concatenated data sets of 4 genes showed that the sequences had high levels of identity to that of the C. siamense strains. According to both morphological and sequence analyses, the pathogen was identified as C. siamense. There were reports of anthracnose on yam caused by Colletotrichum sp. in Guangxi (Zhu et al. 2007), Hainan (Lin et al. 2018) and Jiangsu (Han et al. 2020) provinces in China. To our knowledge, this is the first report of anthracnose on D. opposita caused by C. siamense in Guangdong province in China.